Delta-tocopherol



Patented Nov. 1, 1949 UNITED STATES PATENT OFFICE DELTA-TOCOPHEROL James G. Baxter, Max H. Stern, and Leonard Weisler, Rochester, N. Y.,' assignors to Distillation Products, Inc., Rochester, N. poration of Delaware Y., a cor- No Drawing. Application July 19, 1946, Serial No. 684,770

9 Claims. (Cl. 260-333) gamma-tocopherol were the only three natural tocopherols known. We have isolated this new tocopherol from soybean oil.

This invention has for its object to provide a compound useful as an antioxidant. Another object is to provide a procedure for isolating said compound. A further object is to provide for the conversion of delta-tocopherol in a soybean oil concentrate to other tocopherols, in particular to alpha-tocopherol. A still further object is to improve the state of the art. Other objects will appear hereinafter.

These and other objects are accomplished by our invention which includes concentrating deltatocopherol from vegetable oils by procedures such as high-vacuum, unobstructed-path distillation, saponifioation, chromatographic absorption, solvent extraction and/or esterification. The material can be converted into alpha-tocopherol, preferably after the purification step.

Reference is made to Weisler application Serial No. 684,769 of this date covering the conversion to alpha-tocopherol of delta-tocopherol and also betaand gamma-tocopherol as well as tocopherol mixtures.

In the following description we have set forth several of the preferred embodiments of our invention but it is to be understood that they are given by way of illustration and not in limitation thereof.

Each of the alpha-, beta-, and gamma-tocopherols has certain distinctive properties such as its activity in the cure of resorption sterility, muscular dystrophey, and as a sparing agent in the body for other vitamins and hormones. This sparing action, also known as co-vitamin action; depends principally on the property of behaving as an antioxidant in the body. The accepted formulas of alpha-, beta-, and gamma-tocopherol are as follows:

CH3 CH2 CH3 CH2 HO E H C-OH; C-0Ea Hi0 H H3 0 CiaHzs CH3 0 On a: Alpha-tocopherol Beta-tocopherol CH; HO

C-GH;

Gnmma-tocopherol The newly discovered natural delta-tocopherol 2 has the following formula according to the evidence available at present.

H3 H8 0 CioHaa Delta-tocopherol It has been isolated from soybean oil. It is less active than alpha-tocopherol in the resorption sterility test but can readily be converted to alpha-tocopherol by synthesis. It is an active antioxidant, and in'particular is an antioxidant or sparing agent for vitamin A.

A characteristic property of delta-tocopherol is its behavior on oxidation. Tocopherols are commonly assayed by the Emmerie-Engel reaction which consists in the formation of a red color by the interaction of tocopherols, ferric chloride and alpha dipyridyl. Substantially complete color formation occurs quickly with alpha-, beta-, and gamma-tocopherols (within 2 minutes) and the color intensity at 10 minutes reaction time differs by 5% or less from the 2-minute value. With delta-tocopherol complete color formation does not occur so quickly and the percentage increase in color between 2 and 10 minutes is about 30%. This percentage increase in color in the Emmerie- Engel reaction between 2 and 10 minutes will be referred to as the percentage rise and is the basis for determining delta-tocopherol in the mixture of tocopherols which accompany it during the purification stages.

The tocopherols in soybean oil consist of approximately 30% delta-tocopherol. This fraction, together with any gamma-tocopherol present, may be converted to alpha-tocopherol by halomethylation followed by reduction.

Examples of suitable methods for separating the tocopherols from soybean oil are highvacuum, unobstructed-path distillation, selective adsorption, solvent extraction, saponification, and combinations thereof. In connection with highvacuum distillation, it is preferred, but not necessary, for the oil to be first refined in order to remove lecithin. The lecithin removal step is well known in the art and does not form a part of our invention. Selective adsorption involves solution of the soybean oil in a solvent having relatively low eluting power and passage of the solution through a column containing an adsorbent, such as Special Filtrol, silica gel, Doucil (a sodium aluminum silicate used as a water softener) aluminum, calcium and magnesium oxides. While we prefer to use solvents of low eluting power, more active solvents can be used, especially with the stronger adsorbents. Solvents with slight eluting activity include petroleum ether and carbon tetrachloride. Examples of so]- vents having higher eluting power are benzene, ether, methanol and acetone. After the tocopherols have been adsorbed on the adsorbent, the solumn is washed with the same or another solvent in order to elute the adsorbed tocopherols therefrom. Usually a stronger eluting solvent in this step.

The adsorption process described has the distinct advantage that it can be very economically carried out in conjunction with a process for solvent extraction of the oil from soybeans. Thus, a hexane solution of soybean oil extract, aszit is obtained in commercial extraction plants, can be directly passed through the adsorbent in the manner described. This would mean that a distillation operation to vaporize the hexane from the extracted oil prior to adsorption would be avoided. Adsorption also has the advantage that unsaturated glyceride constituents in the soybean oil are preferentially adsorbed and can be separated during the adsorption step, thus giving a secondary product having improved drying properties. Gammaand delta-tocopherols are present in soybean oil in about a 2:1 ratio. The gammadeltafraction is much more readily adsorbed than alphaor beta-tocopherol and, since about 90% of the tocopherols in soybean oil are a gammadelta-tocopherol mixture (60% gammaand delta-tocopherol), this procedure is very effective for the recovery of the tocopherol content of soybean oil.

The high-vacuum, unobstructed-path distillation operation is carried out in the conventional manner. Gravity flow or centrifugal force highvacuum unobstructed-path stills may be used. 1

The pressure is that which is conventionally used for such distillations, i. e., below 1 mm., and preferably below 0.1 mm., such as, for instance, 0.01 to 0.001 mm. The temperature is preferably between about 150 and 250 C. The tocopherol content of the soybean oil distills as a preliminary fraction and we prefer to separate a narrow out, since distillation of a large amount of the oil requires extensive exposure to heat and destroys the tocopherol content. A cut of about 1% or less seems to be the optimum amount to be removed as a tocopherol fraction.

The solvent extraction can be accomplished by treating the soybean oil with a solvent which is substantially immiscible therewith at the temperature of extraction, followed by stratification to form a solvent layer and an oil layer, followed by removal of the solvent layer and vaporization of the solvent therefrom, preferably under vacuum to leave a tocopherol concentrate as a residue. Examples of suitable solvents are methyl and ethyl alcohol and furfural. If desired, the mixing of the solvent and oil can take place at an elevated temperature at which the two are more or less miscible, followed by cooling to stratify into two layers.

The concentrate prepared by any of the fore going or other methods is then converted into alpha-tocopherol. This conversion may be directly applied to the product as obtained from the concentration process or further purification steps may be applied thereto. The conversion is effected by introducing methyl groups into the five position of gamma-tocopherol, and into the five and seven positions of delta-tocopherol. The tocopherol mixture may, for instance, be treated with an aqueous solution of formaldehyde in the presence of hydrogen chloride. This results in the introduction of chloromethyl groups into the five position of gamma-tocopherol and thefive and seven positions of delta-tocopherol. These chloromethyl groups are then converted into methyl groups by a reduction procedure, such as by'the Clemmensen procedure, catalytic hydrogenation, or zinc dust and hydrochloric acid. Other hydrogen halides than hydrogen chloride may be used and, instead of using formalin, a polymer, such as paraformaldehyde, may be used. chloromethyl ether may also be substituted for formalin.

After the tocopherols have been concentrated they may be separated from one another by fractional crystallization and chromatography.

In the step consisting of fractional crystallization a mixture of tocopherols is esterified with an acid halide such as palmitoyl chloride. Lauroyl bromide, myristoyl iodide, dinitrobenzoyl chloride, and phenylazobenzoyl chloride are examples of other acid halides which may be used to esterify the tocopherol mixture. These all give alpha-, beta-, gamma-, and delta-tocopherol esters of difiering solubility. The alpha-, beta-, and gamma-esters are precipitated, leaving the delta-esters in solution.

Chromatography may be used to accomplish the desired further concentration of delta-tocopherol. Thus, the mixed tocopherols may be chromatographed on a mild adsorbent, such as Doucil, and developed with a weakly eluting solvent, such as petroleum ether. On eluting the adsorbent, in portions, e. g. with methyl alcoholacetone mixture, delta-tocopherol is concentrated in the portions of adsorbents taken from the upper part of the column. Alpha-, beta-, and gamma-, are concentrated in the portions taken from the lower part of the column. In this manner a solution already rich in tocopherol is chromatographed on a comparatively small column. If a solution of low concentration is used a large column is required and much time is consumed in chromatographing.

Example 1.Concentration of tocopherols by high-vacuum unobstructed-path distillation Two tank cars of crude soybean oil were degummed in known manner byagitating at about C. with 2% water to remove phosphatides, the oil being separated from the phosphatides sludge and gum by centrifuging. After the refining operation the oil contained 0.185% tocopherols. This oil was passed through a degasser at about C. at a pressure of about 1 mm. in order to remove moisture and absorbed gases and then was passed over a centrifugal vaporizing surface of a high-vacuum unobstructed-path still which was at a temperature of approximately C. and pressure of approximately 3 to 15 microns. Two percent, by weight, of the soybean oil was removed as a distillate and analysis indicated that the potency of the distillate was 94 mgs. of tocopherol per gram. Repeating the distillation would effect a corresponding increase in potency.

Example 2.Separation of tocopherol from soybean oil by chromatographic adsorption The soybean oil investigated had a tocopherol content of 0.18% (by Emmerie-Engel assay). A sample (3.0 g.) in petroleum ether (50 cc.) was adsorbed on Doucil (50 g.). The column was washed with petroleum ether (200 cc.), 50% petroleum ether-benzene (200 cc.), 10% benzeneether (200 cc.), 50% benzene-ether (200 cc.) and ethyl ether (200 cc.) Each solvent was collected in a separate fraction, five in all being takenas shown in the table below. The tocopherol contents of the fractions are also given.

Table II W ht Percent 01g oco- Fraction grams pherols (2 min (A) DISTILLATION.293 G. ALKALI REFINED BOY- BEAN OIL DISTILLED, 0.22% MIXED TOCOPHEROLS The original soybean oil in this example had an iodine value of 107.3. The iodine values of fractions 1, 2, 3 and 5 were, respectively, 116.2, 132.3, 133.4 and 222.

Example 3.C'oncentratz'on of tocopherol in soybean oil by solvent extraction oil were extracted. Removal of the solvent by vaporization under vacuum left an oil residue containing 1.18% tocopherol. The starting raw oil contained 0.19% tocopherol.

Example 4.Conversion of tocopherol concenirate from soybean oil to alpha-tocopherol 3.3 g. of a tocopherol concentrate prepared by molecular distillation of soybean oil (62% tocopherols by the Emmerie-Engel assay) was dissolved in '75 cc. of ether. Para-formaldehyde (0.4 g.) and zinc chloride (0.4 g.) was added and a vigorous stream of hydrogen chloride gas passed into the suspension for fifteen minutes at room temperature and the mixture allowed to stand over night. The mixture was washed with water, dried and evaporated. The resulting oil assayed for 46% tocopherol and gave a positive chloride test after a sodium fusion. It was then reduced with zinc-mercury amalgam and concentrated hydrochloric acid in ethanol and then vacuum distilled under molecular conditions after separation from the reaction mixture. TWo distillate fractions were obtained which contained 22.5% of the original tocopherol. A portion of the second distillate was treated with succinic anhydride and pyridine and 0.1 g. of alphatocopherol acid succinate was isolated and identified.

Example 5.-The isolation of delta-tocopherol from soybean oil is divided into 3 steps, as follows.

I. Molecular distillation and saponification. After the mixed tocopherols in the soybean oil (approximate composition alp'ha- :gammadeltaz, :60:30) are concentrated by distilling in a molecular still and selecting the fractions richest in tocopherols, the potent fractions may be saponified by adding potassium hydroxide while refluxing in an atmosphere of nitrogen, then cooling and extracting the unsaponifiable portion with ether. Alternatively the oil might be saponified directly. Details of a distillation and saponification are given in Table II. a

(B) SAPONIFIOATION.A DISTILLATE SIMILAR TO FRACTION 1 ABOVE USED Distillate 100.00 2.38 Non-Sap 10.82 17.9

II. Chromatography. The unsaponiflable fraction with the properties shown in Table 11-3 was desterolated by dissolving in acetone, cooling the solution to -20 C., and filtering. It was then chromatographed on Doucil from petroleum ether solution and developed with petroleum ether. The column was cut into two sections, an upper yellow-orange band and a lower light yellow band. This latter band was eluted with ether-acetone and the solvent removed. The material in this fraction was shown by preliminary chromatography to contain the majority of the substance designated as delta-tocopherol and was further purified by another adsorption step.

The readsorption was performed on zinc carbonate-Celite (70:30 mixture). The adsorption was carried out from petroleum ether solution and development was with petroleum ether. The column was then cut up into seven zones, difierentiated from one another by their fluorescence under ultra violet light (Table III). Fraction 5 was richest in delta-tocopherol as determined by the percentage increase in the Emmerie-Engel assay between two and ten minutes rise). The high percentage rise of this fraction was later judged to be due in part to other substances than delta-tocopherol because the rise for the latter in pure form was only 30%. Details of the adsorption are given in Table III.

(ABOVE) ON ZNCOa-CELITE 70:30 MIXTURE, 40 G.

Filtrate 1 Position of band on column, numbered from bottom up.

i Gammatocopherol.

(Celite is a diatomaceous earth supplied by Johns Manville Corporation.)

III. Esterification.--Fraction 5 was esterified' with phenylazobenzoyl chloride as described by Baxter et al. (J. A. C.- S; 65, 923 (1943)). BY crystallization from isopropyl alcohol orange needles of delta-tocopherol phenylazobenzoate were obtained, M. P. ll-42. An analysis of the crystals gave C: 78.96%; H: 8.95% N: 4.67%.

The azo ester was sharply distinguished from gamma-tocopherol phenylazobenzoate. The latter melted at 8384 and a marked melting point depression occurred when the two esters were mixed.

Example 6.Soybean oil containing a mixture of alpha-, gamma-, and delta-tocopherol concentrates was distilled in a cyclic molecular still. The tocopherols and sterols distilled from l50-l70 C. at 0.003 mm. pressure. The distillates were freed of sterols by crystallization from acetone at low temperatures and the desterolated material was redistilled. The distillates were reddish-orange oils containing -40% mixed alpha-, gamma-, delta-tocopherols. The yield of mixed tocopherol concentrates was from -60% of the tocopherols present in the undistilled oils. The impurities consisted of sterols, glycerides, and unknown compounds of a hydrocarbon-like nature.

The distillates were further concentrated and the alphatocopherol separated from the gammadelta-tocopherol mixtures by adsorption on Special Filtrol and Doucil, using the liquid chromatogram method. By adsorption a concentrate containing 87.5% mixed gammadelta-tocopherols in a 2: 1 ratio Was obtained Such a concentrate weighing 145 g. was esterified with palmityl chloride by dissolving the concentrate in 520 ml. of ethylene chloride and 140 ml. of pyridine. added 75 g. of palmityl chloride in 520 m1. of ethylene chloride. After standing twenty hours at room temperature, the mixture was poured into ether and the extract was washed with dilute hydrochloric acid, 10% aqueous potassium carbonate, 0.5 N potassium hydroxide and water. Aft-er distillation of ether the residue (210 g.) was adsorbed with petroleum ether to remove certain dark colored impurities. The fraction eluted with 4 liters of petroleum ether, was evaporated to dryness, leaving 192 g. of a light yellow oil.

This was crystallized from acetone (1500 cc.) at 5 C. overnight. The crystals of gamma-tocopherol palmitate g.) were filtered and the residue was redissolved in acetone and cooled to 5 C. to induce further crystallization. An oily mass separated (40 g.) from which another 20 g. of gamma-palmi-tate was obtained. The filtrate from the first crystallization was evaporated, leaving a concentrate of delta-tocopherol palmitate. This was saponified, giving an unsaponifiable fraction of 60 g., tocopherol by Emmeric- Engel assay). By chromatography it was found that the mixed tocopherols were present in the ratio of 80% deltato 20% gamma-tocopherol, whereas in the initial concentrate the ratio had been 33% deltato 67% gamma-tocopherol.

Example 7.-The essential principle of Example 6 is the esterification of a mixture of deltaand gamma-tocopherols with an acid chloride to give mixed esters such that the gamma-ester is less soluble than the delta and precipitates leaving the delta-ester in solution. Other aliphatic esters such as the laurates and myristates can be used. Certain aromatic acid chlorides, such as dinitrobenzoyl chloride, form well crystallized esters with gamma-tocopherol and can be used.

If the procedure of Example 6 is followed except that g. of lauroyl chloride is substituted for To this solution was slowly the 75 g. of palmityl chloride the results will be satisfactory. Similarly, 67 g. of myristoylchloride may be substituted for the palmityl chloride of Example 6. Comparable molar quantities of still other acid chlorides could be substituted.

Soybean oil is used to a large extent for industrial purposes, such as for paint. The tocopherol contained therein is, therefore, wasted and is, as a matter of fact, harmful for most of the industrial uses. Thus, the tocopherol slows up the drying action of the oil when used in paints. Furthermore, soybean oil is not a good source of tocopherol having primary vitamin E activity, since it contains mostly gammaand deltatocopherol, which have very little vitamin E action. Our improved procedure results in the conversion of a material which was heretofore wasted into a material having at least times the vitamin E potency of the Wasted product. Therefore, the invention greatly increases the amount of vitamin E active material available for medicinal purposes. If the soybean oil is destined for use as a food the converted alpha-tocopherol can be added to the oil from which gammaand deltatocopherol have been removed so that its vitamin E activity is increased.

Properties of pure delta-tocopherol Purified delta-tocopherol was prepared by saponification of the azo ester and extraction of the unsaponifiable matter. It formed a light yellow oil having the following properties.

1. Extinction coefficient:

iit. l-

The corresponding values for the red quinones of alpha-, beta-, and gamma-tocopherols are:

li"... u)

ft. u) 1 2... i

respectively. (b) With nitric acid: A red quinone was formed with an absorption maximum having 6. Activity as an antioxidant: The efiectiveness of delta-tocopherol as an antioxidant for vitamin A alcohol was tested: Crystalline vitamine A was dissolved in olive oil to prepare a mixture having a. potency of 50,500 u./g. To one sample was added delta-tocopherol in 0.1% concentration. The other contained no added antioxidant. The samples were then uniformly exposed to air at 55 C. and the vitamin A content determined at suitable intervals. The percent recovery of vitamin A is given in Table IV.

Table IV Percent Recovcry of Vitamin Sample A hrs. 8 hrs.

Vitamin A (0.1% delta-tocophorol) 78.3 56.4 Vitamin A (no antioxidant added) 12.8

The effectiveness of delta-tocopherol as an antioxidant is strikingly exhibited by these data.

Proof of structure of deZta-tocopherol Evidence that delta-tocopherol is a tocopherol was obtained by chloromethylation followed by reduction. This process, described in copending Weisler application, introduces methyl groups into vacant positions in the benzene ring. Application of this process to delta-tocopherol gave d-alpha-tocopherol, identified through its crystalline acid succinate. The synthesis of alphatocopherol in this way suggested that delta-tocopherol was a lower homolog of alpha-tocoph erol.

Additional evidence of the tocopherol nature of the delta compound was obtained by putting it through a cycle which is characteristic of the tocopherol nucleus. On oxidation with gold chloride delta-tocopherol formed a yellow oil having an absorption maximum in the range characteristic of the tocopherol p-quinones,

Alpha-tocopherol p-quinone has Eff, (265 m )#353 gamma-tocopherol p-quinone ha On reduction with sodium hydrosulfite a hydroquinone was obtained with The tocopherol hydroquinones are characterized by an absorption maximum nearly the same as that of the pure tocopherol. By cyclization of delta-tocopherol hydroquinone in sulfuric acid, delta-tocopherol was regenerated with OH, HO OH, HO CH:

-QH OH CH 0 01511:: OH

2.6 dlmethyl hydroquinone '10 The 2,6 dimethyl hydroquinone was identified by comparing it with an authentic specimen by carbon-hydrogen analysis, infrared spectra, and by the extinction coeflicient of its reaction product with ferric chloride.

For further evidence synthetic delta-tocopherol was prepared from 2-methy1 hydroquinone (toluhydroquinone) and phytol. Its infra red spectrum was the same as that of natural deltatocopherol. Delta-tocopherol does not occur solely in soybean oil. It has been isolated also from wheat germ oil. All fatty oils containing tocopherols which exhibit a 2 to 10 minute rise in the Emmerie-Engel reaction exceeding 5% may contain delta-tocopherol which can be isolated by the processes herein described.

What we claim is:

1. The process of treating a mixture of tocopherols including delta-tocopherol which comprises reacting said tocopherols including deltatocopherol with an acyl halide and thereby esterfying said tocopherols, and thereafter fractionally crystallizing the resulting mixture of esterified tocopherols and thereby separating said delta-tocopherol from said mixture.

2. The process which comprises separating a mixture of tocopherols includin delta-tocopherol from a vegetable oil containing delta-tocopherol together with other tocopherols, treating said mixture with an acyl halide and thereby esterfying said tocopherols including said deltatocopherol, and thereafter fractlonally crystallizing said esterified tocopherols and thereby separating said esterified delta-tocopherol from said other esterified tocopherols.

3. The process which comprises separating a mixture of tocopherols from soybean oil and isolating delta-tocopherol from said separated mixture by esterifying said mixture with an acyl halide and fractlonally crystallizing the esterified materials.

4. The process which comprises esterifying a mixture of tocopherols including delta-tocopherol with an acyl halide, fractlonally crystallizing said esterified mixture and thereby separatin said delta-tocopherol from said mixture, and thereafter converting said delta-tocopherol to alpha-tocopherol.

5. The process which comprises separating a mixture of tocopherols including delta-tocopherol from a vegetable oil containing delta-tocopherol together with other tocopherols, esterifying said mixture with an aliphatic acid halide and fractlonally crystallizin said esterified mixture and thereby separating esterified delta-tocopherol from said other tocopherols.

6. The process of treating a mixture of naturally-occurring tocopherols including delta-tocopherol together with at least one other tocopherol having a single aromatic hydrogen atom in the nucleus which comprises separating a substantial portion of said delta-tocopherol from said mixture containing said other tocopherol and converting said separated delta-tocopherol to alpha-tocopherol.

7. The process which comprises separating a mixture of tocopherols from soybean oil, isolating delta-tocopherol from said separated mixture by esterifying said mixture with an aliphatic acid halide and fractlonally crystallizing the resulting esterified tocopherols, and thereafter converting said isolated delta-tocopherol to alphatocopherol.

.11 12 8. An acyl ester of a substance havingthefol- REFERENCES CITED lowing formula: The following references are of record in the s: file of this patent: H0 5 UNITED STATES PA'IENTS c om Number Name Date CH3 0 u as 2,231,125 Karrer Feb. 11, 1941 9. An aliphatic acid ester vof a substance havgjff gf 35 1 fig, if ing the following formula: 10

OH FOREIGN PATENTS H Number Country Date L 517,401 Great Britain Jan. 29, 1940 15 526,884 Great Britain Sept. 27, 1940 H8 0 OTHER REFERENCES JAMES G. BAXTER. M AX STERN. & gggsins Org. Reactions I, pa e 64, John Wiley LEONARD WEISLER. 

